The NBD Domain of the Cystic Fibrosis Transmembrane Conductance

The NBD1 Domain of the Cystic Fibrosis
Transmembrane Conductance Regulator
Image made using pymol & PDB 1QH3 from Lewis,
EMBO,23,2,283-293,2004
By Eladio Abreu
Cystic Fibrosis
Cystic Fibrosis (CB) is a lethal autosomal
recessive genetic disease
Symptoms:
– mucus build up
– debilitating chronic pulmonary inflammation
and blockage
– excess loss of salts in sweat
– reduced pancreatic enzyme release
Reduced life span
Source: Defective CFTR
Dysfunctional chloride ion transport in
epithelial cells is responsible for the
disease
A defect in the cystic fibrosis
transmembrane conductance regulator
(CFTR) protein leads to the obvious
transport problem
CFTR
The CFTR was first identified in 1989
Sequence analysis predicted that it was
comprised of:
– 2 MSDs
– 2 NBDs
– 1 R-region
Characteristics consistent with members
of the ABC super family of membrane
transporters.
MSD1 MSD2
Thought to satisfy
binding interactions L
of ATP bound to N S
ATP
NBD2 G
G
Q H
NBD1 G NBD2
ATP H
S
L
C
Signature R
Sequence
ATP
Model for The Cystic Fibrosis
Trans-Membrane Regulator, and
Position of NBD1 Within it
NBD1
Image made using pymol,powerpoint & PDB 1QH3 from Lewis,
EMBO,23,2,283-293,2004
NBD1 and its Significance
In most ABC transporters ATP hydrolysis cycles
between the 2 NBDs lead to confirmational
changes in the MSDs and subsiquent channel
gating.
NBDs have several structural motifs that aid in
ATP binding and hydrolysis.
All of the mutations that lead to CF are in NBD1
of the CFTR
Tertiary Structure and Subdomain Composition of
mNBD1
F1-type ATP binding core c
ABC ά-subdomain
β-subdomain
Unique to mNBD1
N
mNBD1
Image made using pymol, powerpoint & PDB 1QH3 from
Lewis, EMBO,23,2,283-293,2004
ATP Binds between helices Hl, H1B, and
H1C
Walker motif A (GSTGSGKTS) extends from the N-
terminal end of H1.
– ATP directly binds:Gly463, Ser466, Gly461, Ser462, and
Lys464 of walker motif A.
– also binds with the nucleotide indirectly by coordinating
Mg+2 . Mg+2 is coordinated by Thr465 of Walker Motif A
Walker motif B (LYLLDSPFG) which extends from the
S7 β-strand of the F1-type ATP binding core provides an
ASP residue which aids in Mg+2 coordination
Glu493 from an adjacent Q-loop contributes toward
Mg+2 coordination.
Phe430 of helix H1C makes an edge to face interaction
with the adenine of ATP.
Position and Mode of
ATP Binding in
mNBD1
Image made using pymol & PDB 1QH3 from Lewis,
EMBO,23,2,283-293,2004
The image above identifies the H-
bonding, ionic and Vander Vaals
interactions responsible for binding
ATP to NBD1
General position of ATP
binding site
mNBD1 Binds ATP but does not
hydrolize it
Structural characteristics Structural Differences in
shared with other ABC NBDs mNBD1 that eliminate
implicated in ATP binding hydrolytic activity
ATP Binding core
– Walker motifs A and B – Catalytic charged residues in
– Q and H loops these motifs replaced by
inactive serine residues.
ATP only has face to edge
ATP stacks against several interaction with a PHE residue as
aromatic residues when bound result to a different torsional
angle
CF mutations are not in positions
involved in ATP hydrolysis
ATP binding site
Image made using pymol & PDB 1QH3 from Lewis,
EMBO,23,2,283-293,2004
Positions of the common CB causative mutations are
highlighted in cyan, the most common (occurring in 90% of
CB patients) is the deletion of Phe508 (highlighted in green)
Conclusions
NBD1 binds ATP but does not hydrolyze it.
CFTR channel gating is dependent on
ATP binding at NBD1 but not its
hydrolysis.
The NBDS come together to form a
nucleotide sandwich, below the MSDs (as
seen in slide number 5).